The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. This relocation of the H,K-ATPase occurs concomitantly with extensive remodeling of the actin cytoskeleton at the apical membrane, which is also an essential step in the activation of acid secretion. While these aspects of parietal cell activation are well defined, the molecular mechanisms that couple the PKA signaling cascade to mobilization of H,K-ATPases and cytoskeletal remodeling are not known. A coupling protein is ezrin, an 80 kDa phosphoprotein, whose phosphorylation at Ser66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. The long-term goal of our research is to delineate how ezrin orchestrates stimulus-coupled acid secretion. To address this question, three Specific Aims are proposed: first, we will evaluate how phospho-ezrin interacts with ACAP4 using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-ACAP4 contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-ACAP4 interactions in in vitro binding assays. The function of this interaction will then be evaluated by the effects of the peptides on acid secretion using permeabilized gastric glands. Second, we will define the role of ARF6-ACAP4-ezrin apical signaling complex in parietal cell activation. The importance of such an interaction in acid secretion will then be evaluated by functional assay and supra-resolution imaging analysis. Third, we plan to illustrate the spatiotemporal dynamics of SNARE assembly during parietal cell acid secretion. These studies will be facilitated by biochemical and functional characterization coupled with optical imaging in live cells. PUBLIC HEALTH RELEVANCE: Studying the molecular mechanisms underlying parietal cell activation is of great significance in understanding the cellular physiology of regulated epithelial secretion in the gut, and is also expected to be of great benefit in leading to pharmacological strategies for correcting abnormal gastric acid secretion in disorders such as peptic ulcers, and gastroesophageal reflux disease.